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猪柠檬酸合酶与猪线粒体苹果酸脱氢酶融合蛋白中草酰乙酸的静电通道作用

Electrostatic channeling of oxaloacetate in a fusion protein of porcine citrate synthase and porcine mitochondrial malate dehydrogenase.

作者信息

Shatalin K, Lebreton S, Rault-Leonardon M, Vélot C, Srere P A

机构信息

Pre-Clinical Science Unit, Dallas VA Medical Center, University of Texas Southwestern Medical Center at Dallas 75216, USA.

出版信息

Biochemistry. 1999 Jan 19;38(3):881-9. doi: 10.1021/bi982195h.

Abstract

Mitochondrial malate dehydrogenase and citrate synthase are sequential enzymes in the Krebs tricarboxylic acid cycle. We have shown [Lindbladh, C., Rault, M., Hagglund, C., Small, W. C., Mosbach, K., Bülow, L., Evans, C., and Srere, P.A (1994) Biochemistry 33, 11692-11698] that a fusion protein of yeast mitochondrial citrate synthase and yeast mitochondrial malate dehydrogenase channels oxaloacetate between the active sites. A Brownian dynamics simulation model of porcine mitochondrial enzymes of citrate synthase and malate dehydrogenase was used [Elcock, A. H., and McCammon, A. M. (1996) Biochemistry 35, 12652-12658], showing that a positive electrostatic surface potential between the active sites of the fusion protein could account for the channeling of oxaloacetate we observed with the yeast fusion protein. Since the data were established with a yeast fusion protein and the model was with porcine fusion protein, we have now prepared and studied the porcine fusion protein. The channeling of the oxaloacetate intermediate was the same for the porcine fusion protein as it was for the yeast fusion protein. This channeling behavior is eliminated at high ionic strength. A fusion protein of porcine citrate synthase and porcine cytosolic malate dehydrogenase does not exhibit any channeling of oxaloacetate. A model of the fusion protein with the cytosolic malate dehydrogenase shows no clear positive electrostatic potential surface between the two active sites, thus distinguishing it from the fusion protein with the mitochondrial malate dehydrogenase. These results establish the electrostatic nature of channeling in mitochondrial fusion proteins.

摘要

线粒体苹果酸脱氢酶和柠檬酸合酶是三羧酸循环中的顺序酶。我们已经证明[林德布拉德,C.,劳尔特,M.,哈格隆德,C.,斯莫尔,W.C.,莫斯巴赫,K.,比洛,L.,埃文斯,C.,和斯特雷尔,P.A(1994年)《生物化学》33卷,11692 - 11698页],酵母线粒体柠檬酸合酶和酵母线粒体苹果酸脱氢酶的融合蛋白可在活性位点之间传递草酰乙酸。使用了猪线粒体柠檬酸合酶和苹果酸脱氢酶的布朗动力学模拟模型[埃尔科克,A.H.,和麦卡蒙,A.M.(1996年)《生物化学》35卷,12652 - 12658页],结果表明融合蛋白活性位点之间的正静电表面电位可以解释我们在酵母融合蛋白中观察到的草酰乙酸的通道化现象。由于数据是用酵母融合蛋白建立的,而模型是关于猪融合蛋白的,我们现在制备并研究了猪融合蛋白。猪融合蛋白中草酰乙酸中间体的通道化情况与酵母融合蛋白相同。在高离子强度下这种通道化行为会消失。猪柠檬酸合酶和猪胞质苹果酸脱氢酶的融合蛋白不表现出任何草酰乙酸的通道化现象。含有胞质苹果酸脱氢酶的融合蛋白模型显示两个活性位点之间没有明显的正静电表面电位,因此将其与含有线粒体苹果酸脱氢酶的融合蛋白区分开来。这些结果确定了线粒体融合蛋白中通道化的静电性质。

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